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Mini-F plasmid gene that couple host cell division to plasmid proliferation

Authors:

Abstract

A mechanism for stable maintenance of plasmids, besides the replication and partition mechanisms, has been found to be specified by genes of a mini-F plasmid. An oriC plasmid carrying both a mini-F segment necessary for partition [coordinates 46.4-49.4 kilobase pairs (kb) on the F map] and another segment (42.9-43.6 kb), designated ccd (coupled cell division), is more stably maintained than are oriC plasmids carrying only the partition segment; the stability is comparable to that of the parental mini-F plasmid. When replication of a plasmid carrying ccd is prevented and the plasmid copy number decreases, to as few as one per cell, host cell division is inhibited, but not increase of turbidity or chromosome replication. Appearance of plasmid-free segregants is therefore effectively prevented under such conditions. Experimental results suggest that reduction of the copy number of plasmids carrying the ccd region causes an inhibition of cell division and that the ccd region can be dissected into two functional regions; one (ccdB) inhibits cell division and the other (ccdA) releases the inhibition. The interplay of the ccdA and ccdB genes promotes stable plasmid maintenance by coupling host cell division to plasmid proliferation.
Proc.
Natl.
Acad.
Sci.
USA
Vol.
80,
pp.
4784-4788,
August
1983
Genetics
Mini-F
plasmid
genes
that
couple
host
cell
division
to
plasmid
proliferation
(stable
maintenance
of
plasmid/plasmid
partition/host-plasmid
interaction/oriC
plasmid/SOS-like
function)
TERU
OGURA*
AND
SOTA
HIRAGA
Institute
for
Virus
Research,
Kyoto
University,
Kyoto
606,
Japan
Communicated
by
Charles
Yanofsky,
April
11,
1983
ABSTRACT
A
mechanism
for
stable
maintenance
of
plas-
mids,
besides
the
replication
and
partition
mechanisms,
has
been
found
to
be
specified
by
genes
of
a
mini-F
plasmid.
An
oriC
plas-
mid
carrying
both
a
mini-F
segment
necessary
for
partition
[co-
ordinates
46.4-49.4
kilobase
pairs
(kb)
on
the
F
map]
and
another
segment
(42.9-43.6
kb),
designated
ccd
(coupled
cell
division),
is
more
stably
maintained
than
are
oriC
plasmids
carrying
only
the
partition
segment;
the
stability
is
comparable
to
that
of
the pa-
rental
mini-F
plasmid.
When
replication
of
a
plasmid
carrying
ccd
is
prevented
and
the
plasmid
copy
number
decreases,
to
as
few
as
one
per
cell,
host
cell
division
is
inhibited,
but
not
increase
of
turbidity
or
chromosome
replication.
Appearance
of
plasmid-free
segregants
is
therefore
effectively
prevented
under
such
condi-
tions.
Experimental
results
suggest
that
reduction
of
the
copy
number
of
plasmids
carrying
the
ccd
region
causes
an
inhibition
of
cell
division
and
that
the
ccd
region
can be
dissected
into
two
functional
regions;
one
(ccdB)
inhibits
cell
division
and
the
other
(ccdA)
releases
the
inhibition.
The
interplay
of
the
ccdA
and
ccdB
genes
promotes
stable
plasmid
maintenance
by
coupling
host
cell
division
to
plasmid
proliferation.
Plasmids
that
replicate
by
using
the
replication
origin
(oriC)
of
the
Escherichia
coli
chromosome
are
not
stably
maintained
through
cell
division
under
nonselective
conditions
(1).
We
have
previously
found
that
when
a
particular
segment
of
mini-F
plas-
mid
is
inserted
into
such
plasmids,
the
resulting
oriC
plasmids
become
stable
(1,
2).
The
segment
that
contributes
to
this
sta-
bility
has
been
located
within
the
46.4-49.4
kilobase
pairs
(kb)
coordinates
on
the
F
map
(3),
which
is
outside
of
the
region
essential
for
mini-F
replication
(44.0-46.35
kb),
and
the
sta-
bilization
is
achieved
without
a
detectable
increase
in
plasmid
copy
number
(2).
Apparently,
this
segment
specifies
the
par-
tition
rather
than
the
replication
control
of
such
plasmids.
It
has
been
shown
that
the
segment
includes
three
functional
regions
necessary
for
stable
maintenance;
two
(sopA
and
sopB)
act
in
trans
and
one
(sopC)
acts
in
cis
(2)
(Fig.
1).
However,
even
oriC
plasmids
stabilized
by
the
partition
mechanism
of
mini-F
are
not
fully
stable.
This
observation
prompted
us
to
investigate
additional
DNA
segments
for
stabilization
properties.
In
this
paper,
we
describe
the
characterization
of
a
mini-F
DNA
seg-,
ment
that
seems
to
play
an
important
role
in
stable
mainte-
nance
of
plasmids
in
host
bacteria.
This
segment
(42.9-43.6
kb),
designated
ccd
(coupled
cell
division),
is
located outside
of
the
regions
essential
for
autonomous
replication
and
for
par-
tition
of
mini-F
(Fig.
1).
The
ccd
segment
appears
to
act
by
coupling
host
cell
division
to
proliferation
of
plasmids.
We
pro-
pose
a
hypothesis
that
explains
the
functions
of
the
ccd
seg-
ment.
MATERIALS
AND
METHODS
Bacterial
strains
used
were
all
derivatives
of
E.
coli
K-12.
Strains
KY7231
(F-
trpB9578
tna-2.
rpsL
recAl)
and
KZ200
(F-
ilv
thr
metE
trp tyr
thy
rpsL
recAl)
were
our
laboratory
stocks.
Strains
km1213
(F-
polA
his
argG
metB
leu
rpsL
xyl
lacY
thy)
(5)
and
KH802
(F-
met
gal
supE
hsdR)
(6)
have
been
described.
Plas-
mid
pBR322
(7)
was
obtained
from
H.
W.
Boyer;
pSC138
(8),
from
C.
Wada;
pHSG415
(9),
from
T.
Hashimoto-Gotoh;
pKP1033,
from
Takeyoshi
Miki.
Other
plasmid
strains
were
constructed
in
our
laboratory.
Bacterial
cells
were
grown
in
L
broth
(10),
supplemented
when
necessary
with
thymine
at
25
kug/ml.
Plasmid
DNA
was
prepared
as
described
(11-13).
DNA
synthesis
was
measured
by
determining
incorporation
of
[14C]-
thymine
into
trichloroacetic
acid-insoluble
material.
RESULTS
Construction
of
Stable
oriC
Plasmid
pXX299.
Mini-F
plas-
mids
such
as
pSC138
(8)
consist
of
an
EcoRI-generated
f5
frag-
ment
(Fig.
1)
and
a
drug-resistance
fragment
(see
also
Fig.
5),
and
are
stably
maintained
through
cell
division.
Stable
inher-
itance
of
mini-F
plasmids
should
be
due
to
controlled
repli-
cation
and
accurate
partitioning
of
replicated
plasmid
mole-
cules
into
daughter
cells.
We
have
recently
found
that
oriC
plasmids
(e.g.,
pXX258
and
pXXL99
shown
in
Fig.
1)
carrying
the
"C-A2
segment"
of
mini-F
but
lacking
most
of
the
region
necessary
for
autonomous
replication
are
more
stably
main-
tained
than
oriC
plasmids
carrying
only
a
part
of
the
C-A2
seg-
ment
(e.g.,
pXX206
carrying
the
A2
segment
and
pXX230
car-
rying
the
A2
segment
with
a
deletion;
see
Fig.
1)
even
under
nonselective
conditions.
The
C-A2
segment
seems
to
be
re-
sponsible
for
plasmid
partitioning
(2).
However,
even
oriC
plasmids
stabilized
by
the
mini-F
par-
tition
function(s)
(e.g.,
pXX199)
are
not
fully
stable;
strains
car-
rying
such
plasmids
give
segregants
lacking
the
plasmid
at
low
frequencies
(Fig.
2),
unlike
strains
carrying
the
parental
mini-
F
plasmid
pSC138.
This
suggests
that
mini-F
has
another
sta-
bilizing
function
in
addition
to
the
partitioning
mechanism.
If
this
were
the
case,
such
a
function
would
be
specified
by
a
DNA
segment
located
outside
of
the
regions
essential
for
autono-
mous
replication
and
for
partition.
Accordingly,
we
constructed
oriC
plasmid
pXX299
carrying
both
the
partition
segment
and
an
Xho
I
segment
(42.1-44.8
kb)
of
mini-F
(Fig.
1).
This
plas-
mid
was
found
to
be
extremely
stable
under
nonselective
con-
ditions
(Fig.
2).
Similar
results
were
obtained
when
the
plasmid
was
in
another
bacterial
strain,
KY7231.
Thus
the
Xho
I
seg-
Abbreviations:
kb,
kilobase
pairs;
kDa,
kilodalton(s);
Apr,
ampicillin
re-
sistance.
*
Present
address:
Dept.
of
Technology,
National
Inst.
of
Health,
Tokyo
141,
Japan.
4784
The
publication
costs
of
this
article
were
defrayed
in
part
by
page
charge
payment.
This
article
must
therefore
be
hereby
marked
"advertise-
ment"
in
accordance
with
18
U.S.C.
§1734
solely
to
indicate
this
fact.
Proc.
Natl
Acad.
Sci.
USA
80
(1983)
4785
*
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ori
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Apr
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sopA
sopB
sopC
partition
A2
_
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H
D
(440)
FIG.
1.
Physical
and
functional
map
of
EcoRI-generated
f5
fragment
of
F
plasmid
and
structures
of
oriC
plasmids.
The
cleavage
sites
for
re-
striction
endonucleases
are
indicated.
The
numbers
in
parentheses
denote
kb
coordinates
on
the
F
genome
(3).
The
Pst
I
segments
are
called
Al,
D,
B,
C,
and
A2
according
to
Murotsu
et
al.
(4).
The
region
essential
for
autonomous
replication
is
taken
from
Murotsu
et
al.
(4).
ori
denotes
the
replication
origin
of
the
mini-F
plasmid.
The
region
essential
for
plasmid
partitioning
contains
three
sop
genes
(2).
The
ccd
region
is
described
in
this
paper.
Filled
region
on
maps
of
oriC
plasmids,
DNA
derived
from
mini-F
plasmid;
hatched
region,
DNA
from
the
E.
coli
chromosome;
open
region,
the
ampicillin-resistance
(Apr)
segment;
oriC,
replication
origin
of
the
E.
coli
chromosome.
a
denotes
a
spontaneous
deletion,
and
the
num-
ber
in
parentheses
indicates
the
size
of
the
deletion
(base
pairs).
ment
together
with
the
C-A2
segment
seems
to
confer
com-
plete
stabilization
on
oriC
plasmids.
Inhibition
of
Host
Cell
Division
by
Plasmid
Carrying
a
Spe-
cific
Mini-F
Segment.
How
does
the
Xho
I
segment
(42.1-44.8
kb)
stabilize
the
plasmid?
We
assumed
that
the
segment
might
be
responsible
for
coupling
host
cell
division
to
plasmid
pro-
liferation
(see
Discussion).
To
examine
the
possible
effect
of
the
Xho
I
segment
on
cell
division
when
plasmid
replication
is
blocked,
we
constructed
several
pBR322
derivatives
carrying
various
regions
of
mini-F,
and
we
tested
colony-forming
ability
of
polAts
cells
(strain
km1213)
carrying
them
at
420C
(see
col-
umn
I
in
Fig.
3).
Because
replication
of
pBR322,
but
not
mini-
F,
depends
on
DNA
polymerase
I,
which
is
encoded
by
the
polA
gene,
these
plasmids
cannot
replicate
in
poiAM
cells
at
42"C
unless
they
contain
a
mini-F
segment
essential
for
replication.
We
found
that
on
nonselective
L
agar
plates
at
42CC,
cells
car-
rying
pXX306,
pXX312, pXX315,
or
pXX334
form
minute
col-
onies
and
form
fewer
colonies
than
cells
lacking
plasmid.
These
plasmids
all
carry
the
Hpa
I/Pst
I
(42.9-43.6
kb)
segment,
but
not
the
mini-F
origin.
Microscopical
observation
revealed
that
polAts
cells
carrying
these
plasmids
become
much
elongated
upon
incubation
at
420C,
indicating
that
cell
division
is
inhib-
ited.
By
contrast,
cells
carrying
pKP1033
or
pXX304,
which
contains
the
Hpa
I/Pst
I
segment
and
can
replicate
at
420C
by
using
the
replication
origin
of
mini-F,
form
normal-size
colo-
nies
as
well
as
cells
carrying
the
parental
pBR322.
Similarly,
cells
carrying
pXX167,
which
has
neither
the
Hpa
I/Pst
I
seg-
ment
nor
the
mini-F
origin,
also
form
normal-size
colonies
at
420C.
It
is
therefore
concluded
that
when
replication
of
a
plas-
mid
carrying
the
Hpa
I/Pst
I
(42.9-43.6
kb)
segment
of
mini-
*
50
02'
4)
u
S.
m
..!
0
5
10
15
Incubation
time,
hr
FIG.
2.
Stability
of
oriC
plasmids.
Cells
of
E.
coli
KZ200
carrying
an
oriC
plasmid
were
examined
for
plasmid
stability
in
a
nonselective
medium.
Bacteria
were
grown
overnight
at
370C
in
a
selective
medium
containing
ampicillin,
diluted
1:500
to
1:
1,000
with
a
nonselective
me-
dium
(L
broth),
and
incubated
at
3700.
The
culture
was
diluted
at
ap-
propriate
intervals
with
a
prewarmed
fresh
nonselective
medium
to
maintain
exponential
growing
conditions
throughout
the
experiment.
Samples
were
taken
and
diluted,
and
plates
were
incubated
overnight
at
370.
At
least
200
colonies
from
each
sample
were
scored
for
the
pres-
ence
of
the
Apr
plasmid
by
transferring
them
to
selective
agar
plates
containing
ampicillin
by
toothpicks.
Cells
carrying
each
plasmid
showed
no
detectable
differences
in
growth
rate
as
compared
with
plasmid-free
cells.
The
doubling
time
as
determined
by
turbidity
was
50-52
min
in
all
strains
tested.
*,
pXX299;
o,
pXX199;
A,
pXX230.
X
'H
It1
-.
miniF
f5
fragment
.:-
-It
LI
*T
*
-T
H
H H
C4
pXX299
pXX258
oriC
pXXl99
pXX206
oriC
pXX230
or-iC
oriC
.. . .
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pXX304
pXX312
pXX315
pXX306
pXX334
pXX167
pXX256
pXX339
pXX340
pXX335
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(II)
+
+
+
NT
m
NT
m
NT
m
+
m
NT
+
NT
NT
+
+
+
+
NT
_
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H
H
H
H
zu
ts
t~
Cod
to
E
I
I
I
ccdA
ccdB
ccd
FIG.
3.
Structures
of
mini-F
segments
carried
by
pBR322
derivatives
and
properties
of
the
derivatives.
Column
I,
colony-forming
ability
of
km1213
cells
(poiAM)
carrying
the
indicated
plasmids
on
thymine-supplemented
nonselective
L
agar
plates
at
42TC.
+,
Normal-size
colonies;
m,
minute
colonies,
in
reduced
numbers.
Column
II,
suppression
effect
of
the
indicated
plasmid
on
the
inhibition
of
cell
division
that
is
caused
at
420C
by
a
thermosensitive
plasmid
pXX333
carrying
the
ccd
region
(see
text).
KH802
cells
(polAf)
harboring
both
pXX333
and
the
indicated
plasmid
were
transferred
to
4200,
and
samples
were
removed
and
tested
for
viable
cells
as
described
in
the
legend
to
Fig.
4.
+,
The
inhibition
of
cell
division
by
pXX333
was
suppressed
in
the
presence
of
the
indicated
plasmid;
-,
cell
division
was
inhibited
by
pXX333
even
in
the
presence
of
the
indicated
plasmid.
NT,
not
tested.
F,
designated
the
ccd
region,
is
prevented,
host
cell
division
is
inhibited
and
the
cells
cannot
form
normal-size
colonies
on
nonselective
plates.
Kinetics
of
Inhibition
of
Host
Cell
Division.
To
examine
fur-
ther
the
relationship
between
inhibition
of
plasmid
replication
and
that
of
host
cell
division,
the
kinetics
of
increase
of
tur-
bidity,
number
of
viable
cells,
proportion
of
plasmid-carrying
cells,
and
host
chromosomal
DNA
synthesis
were
followed
by
using
polAtS
cells
harboring
pBR322
or
pXX306
carrying
the
ccd
segment.
In
a
strain
carrying
pXX306,
colony-formers
(viable
cells)
continue
to
increase
exponentially
for
3-3.5
hr
(3.5-4
generations)
after
transfer
to
42TC
and
then
stop
increasing
(Fig.
4),
whereas
turbidity
increases
for
at
least
6-7
hr.
Plasmid-free
segregants
appeared
only
infrequently
(about
10%
of
total
col-
ony
formers)
after
7
hr
of
incubation
at
42TC
(Fig.
4).
In
con-
trast,
the
inhibition
of
cell
division
was
not
observed
in
a
strain
carrying
pBR322,
and
segregants
appeared
at
high
frequency
under
the
same
conditions
(about
90%
of
total
colony-formers).
It
should
be
noted
that
the
time
when
colony-formers
stop
in-
creasing
in
the
pXX306-carrying
strain
nearly
coincides
with
the
time
when
plasmid-free
segregants
begin
to
appear
in
pBR322-carrying
cells.
DNA
synthesis
of
the
cells
carrying
pXX306
was
not
inhibited
significantly
as
compared
with
that
of
the
cells
carrying
pBR322
(data
not
shown).
These
results
indicate
that
the
primary
effect
on
host
cell
growth
caused
by
the
replication
inhibition
of
plasmids
carrying
the
ccd
region
is
inhibition
of
cell
division.
It
seems
unlikely
that
the
inhibition
of
plasmid
replication
per
se
inhibits
cell
division,
because
the
strain
carrying
pXX306
grows
normally
and
number
of
colony
formers
increases
for
3-3.5
hr
after
transfer
to
420C
(Fig.
4),
even
though
replication
of
ColE1-type
plasmids
in
the
strain
km1213
(polAtS)
is
inhibited
immediately
after
transfer
to
high
temperature
(5).
The
inhibition
of
cell
division
may
be
caused
by
reduction
of
plasmid
copy
number.
Effect
of
Thermosensitive
pSC1O1
Derivative
Carrying
the
ccd
Segment
on
Host
Cell
Division.
To
confirm
the
effect
of
plasmids
carrying
the
ccd
region
on
cell
division,
we
recloned
the
mini-F
segment
carried
by
the
pXX306
[EcoRI/BamHI
(40.3-40.45
kb)
fragment
and
the
BamHI/Pst
1
(42.85-43.6
kb)
fragment
containing
the
ccd
region;
see
Fig.
3]
onto
a
ther-
mosensitive
pSC101
derivative
vector,
pHSG415
(9),
which
cannot
replicate
at
420C.
The
resulting
thermosensitive
plasmid
pXX333
(structure
not
shown),
which
has
a
chloramphenicol
resistance
gene,
was
introduced
into
a
polA+
strain
(KH802)
(6),
and
cell
division
was
examined
after
transfer
to
420C.
The
in-
crease
of
colony-formers
began
to
be
markedly
prevented
at
2-
2.5
hr
(about
3
generations),
whereas
turbidity
continued
to
in-
crease
for
at
least
4.5
hr.
Plasmid-free
segregants
(chloram-
phenicol-sensitive
cells)
appeared
only
at
low
frequencies
(<5%)
at
4.5
hr
after
transfer.
By
contrast,
no
detectable
inhibition
of
cell
division
was
observed
in
a
parallel
culture
of
KH802
car-
rying
the
parental
pHSG415,
and
segregants
started
to
appear
at
2-2.5
hr,
the
proportion
of
segregants
being
more
than
80%
at
4.5
hr
(data
not
shown).
These
results
are
consistent
with
those
'I
11
I.
Proc.
Natl.
Acad.
Sci.
USA
80
(1983)
I
Proc.
Natl.
Acad.
Sci.
USA
80
(1983)
4787
100
-o
U,
a)
-
U)
e-
cJl
10
1
100
50
10
100
10
C.r
<
so
-
I.
_
:,
°
._
S.
4..
:>,
Wo
C.
1
-1
0
1
2
3
4
5
6
7
Incubation
time,
hr
FIG.
4.
Kinetics
of
cell
growth
and
plasmid
stability
after
transfer
to
420C.
Cells
of
km1213
(po1A')
carrying
pXX306
(Fig.
3)
or
pBR322
were
grown
at
300C
to
a
midlogarithmic
phase
in
a
nonselective
me-
dium
(L
broth
supplemented
with
thymine)
and
transferred
to
42CC.
The
culture
was
diluted
at
intervals
with
a
prewarmed
fresh
nonse-
lective
medium
to
maintain
exponential
growing
conditions.
(A)
Tur-
bidity
of
cultures
was
measured
in
a
Klett-Summerson
calorimeter
with
a
no.
54
filter.
Number
of
colony
formers
was
determined
by
plating
samples
with
appropriate
dilutions
of
cells
onto
L
agar
supplemented
with
thymine.
Colonies
were
scored
after
incubation
overnight
at
300C.
Generation
time
of
both
strains
was
50
min.
(B)
Tetracycline-resistant
clones
carrying
the
plasmid
as
a
percentage
of
total
colony-formers
was
determined
as
described
in
the
legend
to
Fig.
2,
except
that
selective
agar
plates
contained
tetracycline
and
they
were
incubated
at
300C.
Open
symbols,
pXX306;
closed
symbols,
pBR322.
described
above
for
pBR322-derived
plasmids
in
polAt0
cells.
When
a
recAl
polA'
strain
(KZ200)
carrying
pXX333
was
transferred
to
42TC,
colony-formers
stopped
increasing
at
about
the
same
time
that
plasmid-free
segregants
began
to
appear
in
a
parallel
control
culture
of
KZ200
cells
carrying
pHSG415
after
transfer
to
42TC.
This
indicates
that
the
ccd
function
blocking
host
cell
division
is
independent
of
recA+
activity.
Suppression
of
Growth
Inhibition
by
a
Coexisting
Repli-
cation-Proficient
Plasmid
Carrying
the
ccd
Region.
If
we
as-
sume
that
the
reduction
of
copy
number
of
a
ccd-carrying
plas-
mid
induces
inhibition
of
cell
division,
it
might
be
expected
that
the
inhibition
is
suppressed
by
the
presence
of
another
plasmid
that
also
has
the
ccd
segment
and
can
replicate
at
420C
in
the
polAts
cells.
Accordingly,
we
introduced
mini-F
plasmid
pSC138
(see
Fig.
5)
into
the
polAt0
strain
carrying
pXX306
(see
Fig.
3)
and
examined
its
cell
division
after
transfer
to
42TC.
The
cell
division
of
the
transformant
carrying
both
pXX306
and
pSC138
was
no
longer
inhibited
after
transfer
to
high
temper-
ature,
supporting
our
expectation
(data
not
shown).
Similar
suppression
of
the
inhibition
was
observed
in
a
polA+
strain
(KH802)
carrying
thermosensitive
pXX333
in
the
presence
of
pKP1033
or
pXX306,
which
replicates
at
420C
(see
column
II
in
Fig.
3).
In
contrast,
inhibition
of
cell
division
by
pXX333
was
not
suppressed
by
coexisting
pBR322
derivatives
lacking
the
ccd
region
(e.g.,
pXX256;
Fig.
3).
Therefore
the
inhibition
of
cell
division
caused
by
a
nonreplicative
plasmid
carrying
ccd
is
suppressed
by
the
simultaneous
presence
of
a
replicative
plas-
mid
that
also
carries
ccd.
Further
Dissection
of the
ccd
Region.
We
have
constructed
a
pBR322
derivative,
pXX339,
carrying
a
BamHI/Xma
I
(42.85-
43.35
kb)
segment,
which
is
part
of
the
ccd
region,
and
ex-
amined
its
properties.
Although
cell
division
of
poAts
cells
car-
rying
this
plasmid
is
not
inhibited
at
420C
(see
column
I
in
Fig.
3),
the
plasmid
suppresses
the
inhibition
of
cell
division
of
polA'
cells
exerted
by
pXX333
at
420C
(see
column
II
in
Fig.
3).
The
inhibition
of
cell
division
by
pXX333
is
suppressed
also
by
co-
existing
pXX340
but
not
pXX335,
which
carries
the
BamHI/
Acc
I
(42.85-43.0
kb)
segment
(see
column
II
in
Fig.
3).
These
results
suggest
that
the
ccd
region
contains
two
distinct
func-
tional
regions;
one
(ccdA)
specifies
the
suppression
function
and
the
other
(ccdB)
specifies
the
inhibitory
function
for
cell
di-
vision
(see
Discussion).
The
ccdA
gene
seems
to
be